We are investigating LOK specificity in multiple ways. First, to better understand the peptide specificity of LOK, we are investigating a cutting edge peptide array approach. We have initiated pilot studies with LC Sciences, who started offering the service of massively parallel peptide synthesis on a microchip based on microfluidics technology (4000 individual custom peptides up to 11 residues in length on a single chip). We have encountered technical problems with kinase precipitation and low signal. We are working on overcoming these problems by optimization of kinase preparation and buffers. Second, assays of mutated peptide demonstrate that the uniquely strong preference of LOK for tyrosine at P-2 is most evident in short peptides, but is less dominant in longer peptides. Third, we are therefore undertaking mutational analysis of intact moesin and moesin tail construct, to ascertain the extent to which Y at P-2 contributes to phosphorylation of key biologically relevant substrate(s). We have previously reported the structural basis of the well known specificity of many basophilic kinases for R at P-2, which is provided by two key conserved acidic residues in the peptide binding pocket of those kinases. Based on the strong conservation of overall organization of the eukaryotic protein kinase domain we predicted that the analogous pocket in GCK kinases will bind Y at P-2, but the identity of the critical residues in the GCK family will have evolved. The results (Fig.4) demonstrate strong conservation (within each subfamily) of the P-2 binding pocket. In the P-2 pocket of the GCK family the PEN motif is substituted by AGN, thus replacing bulky charged residues by much smaller and neutral residues to accommodate bulky tyrosine. In addition the YEM motif is substituted by IEM (where I is much less bulky than Y). We are testing the hypothesis that single mutations of these critical residues to the corresponding residue in Rho kinase will reduce or destroy the preference for Y at P-2. Our work on the LOK knockout mouse, demonstrated that although LOK is the dominant ERM kinase in lymphocytes, other ERM kinases must exist (to explain the residual phosphorylation observed). Multiple lines of evidence suggest other GCK kinases (ie close structural relatives of LOK) will also be ERM kinases in lymphocytes. We have analyzed a MST1 knockout mouse, and found no consistent decrease in ERM phosphorylation. But the strongest confluence of evidence points to SLK as an ERM kinases. To answer that question definitively, we have initiated generation of a conditional SLK-knockout mouse. We have obtained chimeras and are now initiating breeding.